Method to produce highly pure eicosapentaenoic acid or its ester

ABSTRACT

A mixture of fatty acids or their esters derived from natural oils and fats including eicosapentaenoic acids or their derivatives is precision distilled under a high vacuum using a plurality of distillation columns, three distillation columns or more in particular, in order to acquire a fraction consisting mainly of fatty acids of carbon number 20 or their esters, which is then subjected to a column chromatography with a reversed-phase distribution system. Eicosapentaenoic acids or their esters, which is useful for preventing or treating thrombus, with a purity as high as 99% and above and with a recovery rate 55% or above, is produced.

This application is a 371 of PCT/JP92/01393 filed Oct. 28, 1992.

TECHNICAL FIELD

The present invention relates to a method of producing highly pureeicosapentaenoic acids or their esters. More particularly, the presentinvention relates to a novel, highly efficient method of producinghighly pure eicosapentaenoic acids (EPA) or their esters, which is auseful in the treatment and prevention of thrombotic diseases.

BACKGROUND TECHNOLOGY

It is well known for many years that eicosapentaenoic acids (EPA), theiresters, amides and the like are useful for the prevention of thrombusand the treatment of thrombotic disease.

It is also well known that these eicosapentaenoic acids occur in naturaland aquatic oils and fats, such as in mackerels, sardines, and cods, inparticular, per se or as derivatives such as glycerides. Many publishedpapers propose a method to separate eicosapentaenoic acids from thesefish oils and the like.

Natural fats consisting of these fish oils, however, contain, inaddition to eicosapentaenoic acids of carbon number 20 and double bondnumber 5, an overwhelmingly large quantity of diverse fatty acids withcarbon numbers ranging from 12 to 24, and double bond numbers from 0 to6. It is thus very difficult to effectively isolate and refineeicosapentaenoic acids as a pure product of a high concentration.

As one of the methods to isolate and refine eicosapentaenoic acids, amethod to rectifying a mixture of fatty acids containingeicosapentaenoic acids and then refining them by forming urea adducts isknown (publication of unexamined patent application Nos. 149400-1982 and8037-1983). With this method, however, eicosapentaenoic acids are onlyinsufficiently isolated from other C20 high level unsaturated fattyacids and intervening C18 and C22 high level unsaturated fatty acids,with the result that only 95% pure products are produced at best. Infact, the method disclosed in unexamined patent application No. 149400indicates that a 93.5% purity is the highest achievable level in itsworking example 3. EPA recovery is also a very low 30%.

The use of reversed-phase partition type column chromatography isproposed as a means to partition-refine eicosapentaenoic acids(publication of unexamined patent application No. 88339-1983). With thismethod, however, in spite of using raw materials containing highlyconcentrated (30%) eicosapentaenoic acids, the same quantity ofdocosa-hexaenoic acids (DHA) are included in the final product, andthese two are only incompletely separated, with the result that theyield of 95% pure eicosapentaenoic acids is only 40%.

It is thus very difficult with conventional methods to produce highlypure eicosapentaenoic acids, those of 95% purity and above inparticular, from a mixture of fats with a high recovery rate.

DISCLOSURE OF THE PRESENT INVENTION

The present invention has been made in view of the above circumstancesto provide a novel method, by eliminating the drawbacks of conventionalmethods, to produce highly pure eicosapentaenoic acids or their esterswith a high recovery rate, which is also available for acquiringproducts which are 95% pure or above.

To solve the problems encountered in conventional methods, the presentinvention provides a method to produce highly pure eicosapentaenoicacids or their esters wherein a mixture of fatty acids or their esterproduced from natural oils and fats containing eicosapentaenoic acids ortheir derivatives is precision distilled under a high vacuum using aplurality of distillation columns to derive a fraction mainly comprisingfatty acids of carbon number 20 or their esters, which is then subjectedto a reversed-phase partition type column chromatography for partitionrefinement.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of a continuous distillation method used inthe present invention.

FIG. 2 is the absorbency spectrum of a chromatography as a workingexample of the present invention.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The key points of the present invention are removal to the extentpossible of fatty acids other than C20 fatty acids by aligning thecarbon number of fatty acids or their esters through high levelmulti-column rectification using a plurality of distillation columns ofa high separating power, followed by partition refining by means of theabove-mentioned reversed-phase partition type column chromatography,thereby producing eicosapentaenoic acids or their ester of a super highpurity of 95% and above, 99% and above in particular, in the industrialproduction achieving a dramatically increased recovery rate.

It is totally impossible with conventional methods to acquire productsof such a high purity with a high recovery rate, and the possibility hasnever been envisaged. More specifically, long-chain high levelunsaturated fatty acids such as eicosapentaenoic acids have a greatnumber of double bonds in the molecule, and thus deterioration,polymerization and other thermal degeneration occur easily, making itextremely difficult to concentrate them by distillation. Additionally,natural oils and fats containing eicosapentaenoic acids contain variousother fatty acids whose boiling points are close to each other, so thatit is impossible to separate eicosapentaenoic acids from other fattyacids unless the height of the distillation column and the quantity ofreflux are increased considerably. This results in the increased columnbottoms pressure and thermal degeneration due to the resultanttemperature rise, eventually making it extremely difficult to refineeicosapentaenoic acids by distillation. This exemplifies that it isextremely difficult to acquire eicosapentaenoic acids sufficientlyselectively as high level unsaturated long-chain fatty acids from amixture of fats containing eicosapentaenoic acids.

With the present invention, as described above, rectification is carriedout under a high vacuum using a plurality of distillation columns. Morespecifically, the method is executed using more than one column.Preferably, a mixture of natural oils and fats containingeicosapentaenoic acids or their ester is introduced into three or moredistillation columns, one out of which is an independent rectifyingcolumn for the initial fraction consisting of fatty acids of a lowcarbon number: the bottoms liquids of these distillation columns arereturned to respective distillation columns one stage before; anddistillation is carried out continuously under a reduced pressure of 10Torr or below and at a bottoms temperature of 210° C. or below.

Desirable embodiments include, in the above continuous distillation, afeed of condensate liquids in the overhead fraction in the distillationcolumn one stage before into the above-mentioned rectifying columnassigned to the initial fraction, and provision of two independentrectifying columns for main fraction containing eicosapentaenoic acidsand their ester as major components and for after-fraction (residue),respectively, for sustaining continuous operation.

Another desirable embodiment of the present method is provision of anindependent vacuum and an condensate system for each distillationcolumn.

The continuous distillation method according to the present inventionmay be a packed, spring or shelf type, but is not limited thereto.Preferably, a sieve plate may be used and the number of theoreticalplates may be five or more.

The continuous distillation column used in the present method can berealized under a reduced pressure of 10 Torr or less, preferably about0.1 Torr, and with the bottoms temperature at 210° C. or less,preferably 195° C. or less.

In the case where three or more distillation columns are used, onecolumn is exclusively used as a rectifying column for recovering initialfraction. For example, when three columns are used, the columns areclassified as follows:

(I) First distillation column

(II) Second distillation column (rectifying column for initial fraction)

(III) Third distillation column (rectifying column for main andafter-fraction)

Four columns, when used, are classified as follows:

(I) First distillation column

(II) Second distillation column (rectifying column for initial fraction)

(III) Third distillation column (rectifying column for after-fraction)

(IV) Fourth distillation column (rectifying column for main fraction)

It is also possible to classify as follows when three columns are used:

(I) First distillation column (rectifying column for initial fraction)

(II) Second distillation column (rectifying column for after-fraction)

(III) Third distillation column (rectifying column for main fraction)

It is of course possible to classify the rectifying columns to be usedmore minutely.

In any case in the present method, the bottoms liquids in the rectifyingcolumn for initial fraction are returned as a reflux to the distillationcolumn one stage before, or the first distillation column in the aboveexamples. It is preferable that each column has an exclusive andindependent vacuum system because the overhead fraction in the firstdistillation column is condensed and then fed to the rectifying columnfor initial fraction in the form of condensate liquid and, further, thevacuum level and bottoms temperature must be strictly controlled in eachdistillation column.

The present continuous distillation method is described in more detailreferring to the attached drawings. The example shown in FIG. 1 in whichfour distillation columns are used, a mixture of fatty acids or theirester (A) is continuously distilled in four distillation columns (1)through (4).

Each distillation column (1), (2), (3) or (4) is provided with anindependent vacuum system (5), (6), (7) or (8), a condensing system (9),(10) or (12), and a reboiler (13), (14), (15) or (16).

Distillation columns (1) through (4) are strictly controlled at areduced pressure of 1 Torr or below, and at a bottoms temperature of200° C. or below. Vacuum level and temperature are closely related toeach other and hence it is preferable to provide an independent vacuumsystem for each distillation column, but it is not necessarily essentialto provide vacuum systems (5) through (8) which are completelyindependent from each other for the purpose of the above control. Thevacuum system may be suitably constructed according to the capacity ofvacuum pumps and control systems to be used, etc.

In the above structure, raw materials (A) are fed to the firstdistillation column (1), near the overhead, for example. The overheadfraction is condensed in the condensing system (9) and fed to the seconddistillation column (2) serving as a rectifying column for the initialfraction, at its bottoms, for example. Introduction of the material inthe form of liquid is an important factor.

In the second distillation column (2), initial fraction (B) consistingof fatty acids of a low carbon number (<C10) is withdrawn from theoverhead. A part of the bottoms liquids is returned to the firstdistillation column (1) at the overhead or its vicinity. This is anotherimportant feature of the method according to the present invention.Bottoms condensate liquids in the first distillation column (1) areheated in the reboiler (13) and returned to the bottoms of the columnwhile introduced into the third distillation column (3) at the overheador its vicinity in the form of liquid.

Overhead components in the third distillation column (3) are supplied ascondensate liquids to the fourth distillation column (4) at the bottomsvia the condensing system (11). Bottoms condensate liquids are heated inthe reboiler (15) and returned to the bottoms while after-fraction(residue) (C) consisting mainly of longer-chain fatty acids of C21 andabove than eicosapentaenoic acids or their ester is recovered.

The overhead (distilled) components in the fourth distillation column(4), supplied with condensate liquids from the overhead of the thirddistillation column (3), are condensed in the condensing system (12),and a part of the product is returned to the overhead or its vicinitywhile the main fraction (D) consisting mainly of eicosapentaenoic acidsor their ester are recovered. Bottoms condensate liquids are heated inthe reboiler (16) and returned to the bottoms while partly returned tothe third distillation column (3) at its overhead or its vicinity.

Raw materials (A) may be pre-treated in a flash tank (17) kept at areduced pressure before they are introduced to the first distillationcolumn (1) to remove air, moisture and other impurities. It is alsofavorable to use a falling-film evaporation type reboiler for reboilers(13) through (16), in order to shorten heating time. The use of afalling-film evaporation type reboiler is effective for preventingthermal deterioration as well.

The above continuous distillation method avoids the problems encounteredin conventional methods described above, and enables production ofhighly pure eicosapentaenoic acids or their ester of a highconcentration of 80% and above with a high efficiency and by simpleoperation using only the distillation process for refining.

The mixture of fatty acids or their esters to be processed may bearbitrarily obtained from natural oils and fats containing a largequantity of eicosapentaenoic acids or their glycerides and otherderivatives, such as a mixture of fatty acids or their esters obtainedfrom suitable items such as sardine, mackerel, herring, mackerel pikeand other, fish and marine zooplankton.

When desired, the mixture of these fatty acids is esterified forcontinuous distillation.

In the present invention, materials are introduced to a reversed-phasepartition type column chromatography for partition refining after theabove continuous distillation. In this particular case, alkyl bondsilica packing materials or the like, for example, is used in columns inorder to construct a so-called reversed-phase distribution system.Water, alcohol, ketone or the like may conveniently be used as thesolvent system. These materials are used independently or mixed.

Possible pre-processes for the above chromatography include removal oflow level unsaturated fatty acids or ester by means of urea addition,low temperature separation and the like.

Production efficiency is further improved in the chromatography byremoving beforehand those components which can easily be separated fromeicosapentaenoic acids or by carrying out other suitable operations forthe multi-column system.

Through partition refining with the column chromatography in thisreversed-phase distribution system, the present invention enablesacquisition of eicosapentaenoic acids or their ester of 99% purity andabove at a 55% recovery rate and above.

The production method according to the present invention is described inmore detail referring to working examples below.

Working Example 1

Ethyl ester of a mixture of fatty acids derived from fish oil with thefollowing composition was rectified in a four-column type rectifyingsystem shown in FIG. 1.

    ______________________________________                                               C10 and below  60%                                                            C20            23%                                                            (of which EPA  16.5%)                                                         C21 and above  17%                                                     ______________________________________                                    

More specifically, the above ethyl ester mixture was processed in aflash tank (17) kept at 1 Torr vacuum, and supplied to the firstdistillation column (1), 300 mm in diameter and approximately 7 metershigh, kept at 0.1 Torr vacuum at a rate of 16.4 kg/h.

Bottoms temperature was controlled at 195° C. or below, or moreconcretely at 193° to 195° C., in the first distillation column (1). Thenumber of theoretical plates was four. Vacuum level and temperature atthe bottoms of the first distillation column (1) were difficult tocontrol because a mixture of fatty acid ester of C20 and above waseasily accumulated at the bottoms. To solve the problem, a smallerquantity of packing materials were used in the first distillation columnthen they were used in the second distillation column (2).

Overhead condensate liquids in the first distillation column (1) wereintroduced into the second distillation column (2) at the bottoms.Bottoms temperature in the second column was controlled at 184° to 185°C. The column was operated under a reduced pressure of 0.1 Torr. Thenumber of theoretical plates was six. The overhead fraction was returnedat a reflux ratio of 1:2. A part of the overhead fraction was recoveredat the rate of 9.9 kg/hr as the initial fraction (B).

The initial fraction was composed of 99% fatty acids of C10 and below,1% C20 eicosapentaenoic acid ester, etc. and 0% fatty acids of C21 andabove.

In the second distillation column (2), the bottoms liquids werecontrolled at a constant liquid level, and returned to the firstdistillation column (1) at the overhead or its vicinity. This means thatsaid bottoms condensate liquids were returned to the first reflux column(1) as a reflux. Bottoms liquids in the first distillation column (1)were supplied to the third distillation column (3) at the overhead orits vicinity. The pressure at this time was a reduced pressure of 0.1Torr and the bottoms temperature was controlled at 195° C. or below inlike manner. The number of theoretical plates was four.

Bottoms liquids in the third distillation column (3) were recovered aspost-fraction (residue) (C). The post-fraction was composed of 0.1%fatty acid C10 and below, 18% C20 eicosapentaenoic acid ester, etc., and81.9% fatty acids C21 and above.

Overhead fraction in the third distillation column (3) was supplied tothe fourth distillation column at the bottoms as condensate liquids. Thefourth distillation column (4) had six theoretical plates, and wasoperated under a reduced pressure of 0.1 Torr and at a bottomstemperature of 195° C. or below.

The bottoms liquids were returned to the third distillation column (3)at the overhead as a reflux. In this case, the bottoms liquids in thefourth distillation column were controlled at a constant liquid level.

The overhead condensate liquids were returned at a reflux ratio of 1:2while the main fraction (D) was recovered at a rate of 2.3 kg/h.

The main fraction was composed of 0.1% fatty acids C19 and below, 0%fatty acids C21 and above, and 99.9% C20 eicosapentaenoic acid ester,etc.

Concentration of eicosapentaenoic acid ethyl ester was 82.77% as shownin Table 1.

                  TABLE 1                                                         ______________________________________                                        Ca:b ester composition (a and b indicate carbon number                        and double bond number, respectively)                                         ______________________________________                                        C18:1 ethyl ester   0.09%                                                     C20:1 ethyl ester   7.83%                                                     C20:4 ethyl ester   9.01%                                                     Eicosapentaenoic acid ethyl ester                                                                 82.77%                                                    Other C20 ethyl ester                                                                             0.30%                                                     ______________________________________                                    

Working Example 2

Main fraction produced in Working Example 1 was subjected to high speedliquid column chromatography. Five grams of main fraction was injectedinto a column which was a stainless steel tube 5 cm in diameter and 50cm long packed with octadecyl silica gel of particle size 10 to 20 umand eluted with ethanol of liquid quantity of 75 mi/min. The detectionwas made by 210 nm absorption. The resultant chromatogram is shown inFIG. 2. An eluate from the area corresponding to the hatched area waspicked and the solvent was removed under a reduced pressure with arotary evaporator to produce 3.55 g clear and colorless oily substance.The substance consisted of 99.47% eicosapentaenoic acid ethyl ester and0.54% C20:4 ethyl ester. The recovery rate for eicosapentaenoic acidethyl ester in this process was 85.3%. In contrast, the recovery ratefor eicosapentaenoic acid ethyl ester from the mixed ethyl ester, theraw material used in Working Example 1, was 60%.

Working Example 3

Methanol in Working Example 2 was substituted with a solvent of 50:50methanol:acetone for chromatography partition.

As a result, eicosapentaenoic acid ethyl ester of a purity of 99.56% wasproduced with 61% recovery rate.

Industrial Field of Utilization

The present invention enables the production of highly pureeicosapentaenoic acids or their ester of a purity of 99% and above,which is useful as a prescription and the like for the treatment andprevention of thrombotic disease, at an excellent recovery rate of 55%and above.

We claim:
 1. A method of producing highly pure eicosapentaenoic acids ortheir ester wherein a mixture of fatty acids or their esters derivedfrom natural oils and fats containing eicosapentaenoic acids or theirderivatives is precision distilled in a plurality of distillationcolumns under a high vacuum to acquire a fraction consisting mainly offatty acids of carbon number 20 or their esters, followed by subjectingthe fraction to the column chromatography with a reversed-phasedistribution system for partition refining.
 2. A method of producinghighly pure eicosapentaenoic acids or their esters of claim 1 whereinprecision distillation is carried out under a reduced pressure of 10Torr or below and at a temperature of 210° C. or below using three ormore distillation columns.
 3. A method of producing highly pureeicosapentaenoic acids or their esters of claim 1 wherein partitionrefining is carried out with a reversed-phase distribution columnchromatography in which alkyl bond silica packing materials are charged.4. A method of producing eicosapentaenoic acids or their esters at apurity of 99% and above, which consists of the step of subjecting amixture of fatty acids or their esters derived from natural oils andfats to precision distillation in a plurality of distillation columns ofthree or more under a reduced pressure of 10 Torr or below and atemperature of 210° C. or below to obtain a fraction consisting mainlyof fatty acids of a carbon number of 20 or their esters, followed by thestep of subjecting the fraction to column chromatography with areversed-phase distribution system to partition refine the fatty acidsso as to separate and recover eicosapentaenoic acids or their estersfrom said fraction at a purity of 99% and above.
 5. A method ofproducing eicosapentaenoic acids or their esters according to claim 4,wherein the fatty acid fraction having mainly a carbon number of 20 ortheir esters are partition refined by means of a reversed-phasedistribution column in which electrically charged alkyl bond packingmaterials are employed therein.